P
US8383365B2ActiveUtilityPatentIndex 96

Methods of making FGF-21 mutants comprising non-naturally encoded phenylalanine derivatives

Assignee: AMBRX INCPriority: Mar 30, 2007Filed: Mar 18, 2011Granted: Feb 26, 2013
Est. expiryMar 30, 2027(~0.7 yrs left)· nominal 20-yr term from priority
Inventors:CUJEC THOMAS PMARIANI ROBERTOHAYS PUTNAM ANNA-MARIA AKEEFE WILLIAM MKNUDSEN NICKSKIDMORE NEE HO LILLIANPINKSTAFF JASONKRAYNOV VADIM
A61P 39/00A61P 9/10A61P 43/00A61P 3/08A61P 37/02A61P 3/04A61P 31/04A61P 29/00A61P 3/00A61P 3/10A61P 17/02A61P 1/18A61P 11/00A61K 38/1825A61K 45/06A61K 47/60C07K 19/00A61K 38/00C07K 14/50C12N 15/70C12N 15/09Y02A50/30
96
PatentIndex Score
48
Cited by
720
References
60
Claims

Abstract

Modified FGF-21 polypeptides and uses thereof are provided.

Claims

exact text as granted — not AI-modified
1. A method of making a modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is a phenylalanine derivative and
 is at a position selected from the group consisting of residues 72, 77, 86, 87, 91, 108, 110, 126, 131, and 146 of SEQ ID NO: 1 or the corresponding amino acid position in another human FGF-21 polypeptide; and wherein the non-naturally encoded amino acid is linked to a linker, polymer, or biologically active molecule, the method comprising ribosomally synthesizing a modified human FGF-21 polypeptide, which comprises a non-naturally encoded amino acid, and contacting the modified human FGF-21 polypeptide with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid of the modified human FGF-21 polypeptide. 
 
     
     
       2. The method of  claim 1 , wherein the polymer is a water soluble polymer. 
     
     
       3. The method of  claim 1 , wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. 
     
     
       4. The method of  claim 1 , wherein the non-naturally encoded amino acid comprises a carbonyl moiety and the linker, polymer, or biologically active molecule comprises an aminooxy, a hydrazine, a hydrazide or a semicarbazide moiety. 
     
     
       5. The method of  claim 4 , wherein the aminooxy, hydrazine, hydrazide or semicarbazide moiety is linked to the linker, polymer, or biologically active molecule through an amide linkage. 
     
     
       6. The method of  claim 1 , wherein the non-naturally encoded amino acid comprises an alkyne moiety and the linker, polymer, or biologically active molecule comprises an azide moiety. 
     
     
       7. The method of  claim 1 , wherein the non-naturally encoded amino acid comprises an azide moiety and the linker, polymer, or biologically active molecule comprises an alkyne moiety. 
     
     
       8. The method of  claim 3 , wherein the azide or alkyne moiety is linked to a linker, polymer, or biologically active molecule through an amide linkage. 
     
     
       9. The method of  claim 1 , wherein the polymer comprises poly(ethylene glycol). 
     
     
       10. The method of  claim 9 , wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1kDa and about 100 kDa. 
     
     
       11. The method of  claim 9 , wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer. 
     
     
       12. The method of  claim 2 , wherein the polymer is an oligosaccharide. 
     
     
       13. The method of  claim 1 , wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide without a non-naturally encoded amino acid. 
     
     
       14. The method of  claim 13 , wherein the half-life increased from about 10-fold to about 40-fold. 
     
     
       15. The method of  claim 13 , wherein the half-life increased from about 13-fold to about 30-fold. 
     
     
       16. The method of  claim 13 , wherein the half-life increased from about 13-fold to about 18-fold. 
     
     
       17. The method of  claim 13 , wherein the half-life increased from about 20-fold to about 30-fold. 
     
     
       18. The method of  claim 9 , wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide without a non-naturally encoded amino acid. 
     
     
       19. The method of  claim 18 , wherein the half-life increased from about 10-fold to about 40-fold. 
     
     
       20. The method of  claim 18 , wherein the half-life increased from about 13-fold to about 30-fold. 
     
     
       21. The method of  claim 18 , wherein the half-life increased from about 13-fold to about 18-fold. 
     
     
       22. The method of  claim 18 , wherein the half-life increased from about 20-fold to about 30-fold. 
     
     
       23. The method of  claim 1 , wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide with a non-naturally encoded amino acid. 
     
     
       24. The method of  claim 23 , wherein the half-life increased from about 10-fold to about 40-fold. 
     
     
       25. The method of  claim 23 , wherein the half-life increased from about 13-fold to about 30-fold. 
     
     
       26. The method of  claim 23 , wherein the half-life increased from about 13-fold to about 18-fold. 
     
     
       27. The method of  claim 23 , wherein the half-life increased from about 20-fold to about 30-fold. 
     
     
       28. The method of  claim 9 , wherein the modified human FGF-21 polypeptide comprising a non-naturally encoded amino acid exhibits an increased in vivo half-life over the corresponding modified human FGF-21 polypeptide with a non-naturally encoded amino acid. 
     
     
       29. The method of  claim 28 , wherein the half-life increased from about 10-fold to about 40-fold. 
     
     
       30. The method of  claim 28 , wherein the half-life increased from about 13-fold to about 30-fold. 
     
     
       31. The method of  claim 28 , wherein the half-life increased from about 13-fold to about 18-fold. 
     
     
       32. The method of  claim 28 , wherein the half-life increased from about 20-fold to about 30-fold. 
     
     
       33. The method of  claim 1 , wherein the modified human FGF-21 polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 containing a substitution of an amino acid with a non-naturally encoded amino acid. 
     
     
       34. The method of  claim 1 , wherein the modified human FGF-21 polypeptide comprises SEQ ID NO: 1 containing a substitution of an amino acid with a non-naturally encoded amino acid. 
     
     
       35. The method of  claim 1 , wherein the non-naturally encoded amino acid is substituted at residue 108. 
     
     
       36. The method of  claim 1 , wherein the non-naturally encoded amino acid is selected from the group consisting of a para-substituted phenylalanine, an ortho-substituted phenylalanine, and a meta-substituted phenylalanine. 
     
     
       37. The method of  claim 36 , wherein the para-substituted, ortho-substituted, or meta-substituted phenylalanine comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. 
     
     
       38. The method of  claim 1 , wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine. 
     
     
       39. The method of  claim 1 , wherein the non-naturally encoded amino acid is selected from the group consisting of a para-substituted phenylalanine, an ortho-substituted phenylalanine, and a meta-substituted phenylalanine and wherein the polymer is poly(ethylene glycol). 
     
     
       40. The method of  claim 1 , wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the modified human FGF-21 polypeptide comprises SEQ ID NO:1 containing para-acetyl-L-phenylalanine at residue 108. 
     
     
       41. The method of  claim 1 , which results in a biologically active FGF-21 polypeptide having increased in vivo circulating half-life compared to an unmodified FGF-21 polypeptide having the sequence of SEQ ID NO:1. 
     
     
       42. The method of  claim 9 , wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa. 
     
     
       43. A method of making a polypeptide comprising a non-naturally encoded amino acid, wherein the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; wherein the polypeptide contains a substitution of an amino acid with the non-naturally encoded amino acid at a position selected from the group consisting of the corresponding residues 72, 77, 86, 87, 91, 108, 110, 126, 131, and 146 of SEQ ID NO: 1 or the corresponding amino acid position in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; wherein the polypeptide maintains the biological activity of human FGF-21 polypeptides; and wherein the non-naturally encoded amino acid is a phenylalanine derivative and is linked to a linker, polymer, or biologically active molecule, the method comprising ribosomally synthesizing a polypeptide, which comprises a non-naturally encoded amino acid, and contacting the polypeptide with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid of the polypeptide. 
     
     
       44. The method of  claim 43 , wherein the polymer comprises poly(ethylene glycol). 
     
     
       45. The method of  claim 44 , wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa. 
     
     
       46. The method of  claim 44 , wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer. 
     
     
       47. The method of  claim 44 , wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa. 
     
     
       48. The method of  claim 43 , wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine. 
     
     
       49. The method of  claim 43 , wherein the non-naturally encoded amino acid is substituted at residue 108. 
     
     
       50. The method of  claim 43 , wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine substituted at residue 108. 
     
     
       51. The method of  claim 43 , wherein the polypeptide has a serum half-life that is at least five times the serum half-life of FGF-21 polypeptide consisting of SEQ ID NO: 1. 
     
     
       52. The method of  claim 43 , wherein the polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 
     
     
       53. The method of  claim 52  wherein the polymer comprises poly(ethylene glycol). 
     
     
       54. The method of  claim 53 , wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa. 
     
     
       55. The method of  claim 53 , wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer. 
     
     
       56. The method of  claim 53 , wherein the poly(ethylene glycol) moiety has an average molecular weight of about 30 kDa. 
     
     
       57. The method of  claim 52 , wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine. 
     
     
       58. The method of  claim 52 , wherein the non-naturally encoded amino acid is substituted at residue 108. 
     
     
       59. The method of  claim 52 , wherein the polymer is poly(ethylene glycol) having an average molecular weight of about 30 kDa, wherein the non-naturally encoded amino acid is para-acetyl-L-phenylalanine substituted at residue 108. 
     
     
       60. The method of  claim 42 , wherein the polypeptide has a serum half-life that is at least five times the serum half-life of FGF-21 polypeptide consisting of SEQ ID NO: 1.

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